Water-compatible reducible compounds and their use in analytical compositions and methods

ABSTRACT

Certain water-compatible reducible compounds are useful in analytical compositions and elements for assay of various analytes, e.g. microorganisms. These compounds comprise a moiety which provides a detectable species (e.g. a dye) when released from the compound at physiological pH. Further, these compounds are aromatic derivatives or quinones having water-compatibilizing substituent which allow them to be used in compositions without the use of surfactants.

This is a divisional of application Ser. No. 868,855, filed May 30,1986, now U.S. Pat. No. 4,853,186.

FIELD OF THE INVENTION

This invention relates to clinical chemistry. In particular, it relatesto water-compatible reducible compounds which can be reduced to providea detectable species, and to analytical compositions and elementscontaining same. It also relates to a method for the determination ofanalytes in, e.g. biological fluids.

BACKGROUND OF THE INVENTION

Chemical analysis of liquids, such as water, milk and biological fluidsis often desirable or necessary for health maintenance and diagnosticcare. Various compositions and elements to facilitate such analyses areknown. Such compositions and elements generally include a reagentcomposition for determining a substance under analysis, identified as an"analyte" herein. The analyte can be a living organism or a nonlivingchemical substance. The reagent composition, upon interaction with theanalyte, provides a detectable change (e.g. dye formation).

Recently, much work has been directed to developing compositions andelements which are useful for rapid and highly quantitative diagnosticor clinical analysis of biological fluids such as whole blood, serum,plasma, urine and the like.

For example, for the rapid and effective diagnosis and treatment ofinfectious diseases, it is desirable to be able to detect the bacteriacausing the disease as rapidly as possible. Infections of the urinarytract are among the most common bacterial diseases, second in frequencyonly to infections of the respiratory tract. In fact, in many hospitals,urinary tract infections are the most common form of nosocomialinfections, often following the use of in-dwelling catheters and varioussurgical procedures. Most urinary tract infections (UTI) result fromascending infection by microorganisms introduced through the urethra andvary in severity from an unsuspected infection to a condition of severesystemic disease. Such infections are usually associated with bacterialcounts of 100,000 (10⁵) or more organisms per ml of urine, a conditionreferred to as significant bacteriuria. Under normal conditions, urineis sterile, although contamination from the external genitalia maycontribute up to 1,000 (10³) organisms per ml in properly collected andtransported specimens.

A significant advance in the detection of microorganisms and otheranalytes capable of reducing a reducible compound is described andclaimed in copending and commonly assigned U.S. Ser. No. 824,766, filedJan. 31, 1986 by Belly et al and entitled REDUCIBLE COMPOUNDS ANDANALYTICAL COMPOSITIONS, ELEMENTS AND METHODS UTILIZING SAME. The assaydescribed in that application utilizes reducible compounds which releasea detectable species in the presence of the analyte. While providing ahighly advantageous assay, the reducible compounds described thereingenerally have limited solubility in aqueous solutions. Hence,water-solubilizing surfactants must be used in the practice of the Bellyet al invention to prepare compositions of the reducible compounds.

The use of surfactants in such compositions has serious drawbacks.Surfactants tend to lyse certain cells (e.g. white blood cells) therebymaking it difficult to detect or identify such cells. Hence, there is aneed in the art for a rapid and highly sensitive assay using reduciblecompounds which avoids the use of surfactants.

SUMMARY OF THE INVENTION

The present invention overcomes the problems of the art with the use ofcertain water-compatible reducible compounds of the structureCAR--R¹)_(n) wherein CAR- is a substituted or unsubstituted aromatic orquinone nucleus, R¹ is a moiety which comprises a shiftable detectablespecies, and n is 1 or 2,

provided the reducible compound is capable of being reduced at a pH of 9or less to release the shiftable detectable species and comprises atleast one water-compatibilizing moiety, and

further provided that when R¹ is replaced with H, CAR--H)_(n) has anE_(1/2) of at least about +100 mV when measured in water.

Particularly useful water-compatible reducible compounds are of thestructure CAR-R¹ wherein CAR- is ##STR1##

R¹ is ##STR2##

R² and R⁴ are independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted aryl or an electron withdrawinggroup,

R³ is R¹, hydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted aryl or an electron withdrawing group, provided that atleast one of R², R³ and R⁴ is an electron withdrawing group, or R³ andR⁴, taken together, represent the atoms necessary to complete asubstituted or unsubstituted strained fused carbocyclic ring,

R⁵ is substituted or unsubstituted alkylene of 1 or 2 carbon atoms,

R⁶ is substituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl or substituted or unsubstituted aryl,

Q is carbonyl or thiocarbonyl,

FRAG is a shiftable detectable species which provides a detectablespecies when released from the reducible compound, and

m is 0 or 1,

provided that when R¹ is replaced with H, CAR-H has an E_(1/2) of atleast about +100 mV when measured in water, and

further provided that at least one of R¹, R², R³ or R⁴ comprises atleast one water-compatibilizing moiety.

The present invention also provides an aqueous composition buffered at apH of 9 or less which consists essentially of the water-compatiblereducible compound described above.

Also, a dry analytical element for the determination of an analytecomprises an absorbent carrier material and contains thewater-compatible reducible compound described above.

This invention also provides a method for the determination of ananalyte comprising the steps of:

A. contacting a sample of a liquid suspected of containing an analytewith the water-compatible reducible compound described above, and

B. detecting the detectable species released as a result of the presenceof the analyte.

The present invention provides a means for using reducible compounds inrapid and highly quantitative determinations of analytes, e.g. enzymes,metabolites or living cells (e.g. bacteria, yeast, fungi, white bloodcells, etc.) in liquids at physiological pH (i.e. 9 or less). Thesereducible compounds can be dissolved in coating compositions or solutionassay compositions without the use of surfactants. Therefore, theproblem encountered with the use of surfactants (i.e. adverse affect oncells) is avoided. In addition, it was unexpectedly found that thesensitivity of the assay was improved in the absence of surfactants.This invention also provides a means for releasing chemically orbiologically useful moieties which can be converted into detectablespecies. These advantages have been achieved by incorporatingwater-compatibilizing groups in reducible compounds.

DETAILED DESCRIPTION OF THE INVENTION

The water-compatible reducible compounds useful in the practice of thisinvention are broadly defined as water-compatible organic compoundscontaining a shiftable detectable species which can be reduced atphysiological pH (i.e. 9 or less) to release the shiftable detectablespecies. The term "shiftable" is defined as: (1) a chromogen moiety,which has a first spectral absorption band while attached to thereducible compound and a second spectral absorption band when released,or a fluorogen moiety, which has first spectral excitation and emissionbands while attached to the reducible compound and second spectralexcitation and emission bands when released, (2) a chemically orbiologically useful moiety which is inactive, blocked or otherwiseinaccessible when attached to the reducible compound but active,unblocked or accessible when released, or (3) a chemically orbiologically useful moiety which is active or accessible when attachedto the reducible compound but inactive or otherwise inaccessible whenreleased.

Thus, the detectable species is chemically modified when attached to thereducible compound, e.g. for (1) above the spectral band or bands of thereducible compound are "shifted" from the band or bands that the specieshas when released. Generally, but not necessarily, the band or bands arerelocated to substantially shorter wavelengths when the species is apart of the reducible compound. In all cases, the bands do not overlapto a significant extent. The change (i.e. "shift") from one spectralband to another can be due to the mere release of the moiety from thereducible compound, or alternatively, it can be caused by such releasecoupled with either interaction of the released moiety with metal ionsor a mordant, or coupled with a change in the assay environment (e.g.change in pH). With any such change in the environment, the pH mustremain at 9 or less.

Also, as noted above, the shiftable detectable species can also be achemically or biologically useful moiety which, when attached to thewater-compatible reducible compound, is inactive or blocked or otherwiseinaccessible, but when released at physiological pH becomes biologicallyor chemically active or accessible for further interaction. Thereleased, active species can be detectable itself or is capable of oneor more subsequent chemical, physical or biological reactions to providea detectable species. The method of this invention provides a means forreleasing such moieties, e.g. electron transfer agents, enzymes, enzymesubstrates, enzymes inhibitors, cofactors, catalysts, reactants, etc.upon reduction of the reducible compound, preferably at physiologicalpH, for a variety of chemical or biological purposes.

Further, a shiftable detectable species can be a chemically orbiologically useful moiety which, when attached to the reduciblecompound, is active, or otherwise accessible for one or more subsequentchemical, physical or biological reactions, but when released atphysiological pH becomes inactive or otherwise inaccessible for suchreactions.

The reducible compounds described herein are water-compatible, which isdefined as being more readily dissolvable (or soluble) in polar organicsolvents (e.g. alcohols, acetonitrile, N,N-dimethylformamide,dimethylsulfoxide, etc.), water or aqueous solutions containing a minoramount of one or more polar organic solvents, than in nonpolar organicsolvents. Which solvents are polar and nonpolar is readily determinableby one of ordinary skill in the art. This water-compatibility isimparted by one or more water-compatibilizing substituents on thecompound. Such substituents are broadly defined as moieties which have ahydrophobic parameter (P_(i)) less than about -2.0. Such a parameter isa standard value for a given moiety as described, for example, inQuantitative Drug Design by Y. Martin, Marcel Dekker, Inc., New York,1978. These moieties are either readily ionizable in water (e.g. carboxyor sulfo) or nonionizable in water (e.g. iodoxy or glucosyl). Apreferred water-compatibilizing substituent is carboxy. Other usefulsubstituents include hydroxy, quaternary ammonium, sulfonamido, etc. Thesubstituents can be placed on the CAR- portion of the molecule or on theR¹ portion, or on both. Examples of such compounds and the placement ofthe water-compatibilizing substituents are provided below in Table Iwithout intending to limit the scope of this invention.

More particularly, the compounds useful in this invention have thestructure CAR--R¹)_(n) wherein CAR- represents a substituted orunsubstituted aromatic or quinone nucleus, R¹ is a moiety comprising ashiftable detectable species defined below, and n is 1 or 2. Examples ofsuch nuclei are presented below. Further, when R¹ is replaced by H,CAR--H)_(n) has a reduction potential (E_(1/2)) of at least about +100mV when measured in water. This E_(1/2) value facilitates the reductionand subsequent release of the shiftable detectable species from thecompound at physiological pH (i.e. 9 or less). Such measurements aremade according to standard electrochemical techniques using eitherdifferential pulse polarography or cyclic voltametry (see, e.g. Sawyerand Roberts, Jr., Experimental Electrochemistry for Chemists, John Wiley& Sons, New York, 1974). Preferably, the E_(1/2) is from about +100 mVto about +400 mV as measured in water. Further details of measuring theE_(1/2) are described below prior to Table I. The desired E_(1/2) isachieved by appropriate electron withdrawing groups on the CAR- nucleus,or by a strained fused ring attached to the nucleus or by a combinationof both.

In one embodiment, the water-compatible reducible compounds can bereduced to provide a detectable species through quinonemethideformation, similar to the description by Van de Sande in Angew. Chem.Int. Ed. Engl. 22, pp. 191-209 (1983) and U.S. Pat. No. 4,232,107(issued Nov. 4, 1980 to Janssens), but which have the desired E_(1/2)properties and one or more water-compatibilizing groups.

In another embodiment, useful water-compatible reducible compoundsinclude sulfilimides and sulfenylsulfonamides similar to those describedon page 206 of the Van de Sande reference noted above, but which havethe desired E_(1/2) properties and one or more water-compatibilizinggroups.

In a preferred embodiment, the water-compatible reducible compounds ofthis invention are RIND compounds, i.e. reducible compounds capable ofundergoing intramolecular nucleophilic displacement at physiological pHto release one or more shiftable detectable species when a nucleophilicgroup is generated by at least a one electron reduction of the compound.In other words, such displacement occurs when the RIND compound isreduced by a suitable reductant which provides one or more electrons(described in more detail below). The distinction of these RINDcompounds over the many similar benzoquinone compounds used in thephotographic art is that the RIND compounds have a higher E_(1/2) value,thereby facilitating their reduction and subsequent release of ashiftable detectable species (e.g. a dye) at physiological pH (i.e. 9 orless).

The term "intramolecular nucleophilic displacement" refers to a reactionin which a nucleophilic center on a molecule reacts at another site inthe molecule, which site is an electrophilic center, to effectdisplacement of a group or atom attached to the electrophilic center.Generally, the RIND compounds useful in this invention have thenucleophilic and electrophilic groups juxtaposed in thethree-dimensional configuration of the molecule in close proximitywhereby the intramolecular reaction can take place and a ring is formedhaving from 4 to 7 atoms, and preferably having 5 or 6 atoms.

Particularly useful water-compatible RIND compounds are those which havethe structure CAR-R¹ wherein CAR- is ##STR3##

R¹ is ##STR4## wherein m is 0 or 1, and preferably 1. R⁵ is substitutedor unsubstituted alkylene, preferably of 1 or 2 carbon atoms in thebackbone (e.g. methylene, ethylene, alkoxymethylene, etc.). Mostpreferably, R⁵ is methylene. Q is carbonyl or thiocarbonyl andpreferably carbonyl.

R⁶ is substituted or unsubstituted alkyl preferably of 1 to 10 carbonatoms (e.g. methyl, ethyl, n-propyl, isopropyl, t-butyl, hexyl, decyl,benzyl, etc.), substituted or unsubstituted cycloalkyl preferably of 4to 12 carbon atoms (e.g. cyclobutyl, cyclohexyl, 4-methylcyclohexyl,etc.), or substituted or unsubstituted aryl of 6 to 12 carbon atoms(e.g. phenyl, xylyl, naphthyl, p-nitrophenyl, p-t-butoxyphenyl, etc.).Preferably, R⁶ is lower alkyl of 1 to 3 carbon atoms (substituted orunsubstituted), and more preferably, it is methyl.

FRAG is a shiftable detectable species as defined above. Preferably,along with the remainder of the molecule, it has a first spectral band,but when it is cleaved from the RIND compound, it provides a detectablespecies having a second spectral band. This species is released in anamount which can be directly related to the amount of reductant present.The specific composition of FRAG can vary considerably depending uponthe type of detectable species desired and upon the particular detectionmeans employed.

The shiftable detectable species can be a material which is directlydetectable by a suitable means, as well as a material which can reactwith other substances, e.g. analytes, enzymes or other reagents toprovide a detectable species. Such species include those detectable byradiometric means, including chromogens (e.g. dyes or pigments) whichcan be detected colorimetrically and fluorogens (e.g. fluoroscent dyesor probes) which can be detected fluorometrically. Additionally, thedetectable species can be a phosphorescent species, a chemiluminescentspecies, or any other detectable species known to one skilled in theart.

Particularly useful shiftable detectable moieties are chromogens andfluorogens having a first spectral band(s) prior to release and a secondspectral band(s) when measured after release. Examples of useful classesof chromogens are azo, azomethine, nitrophenol, indophenol, indoanilineand triarylmethane dyes, and others known in the art, with azo dyesbeing preferred. Examples of useful classes of fluorogens are coumarins,4-oxo-4H-benz-[d,e]anthracene and its derivatives, phenalenones andbenzphenalenones, fluorescein and rhodamine fluorescent dyes, and othersknown in the art. Phenalenone dyes are particularly useful.

Useful phosphorescent species include such phosphors as2',5'-dibromofluorescein and 4',5'-diiodofluorescein. A usefulchemiluminescent species is luciferin.

FRAG is linked to Q by means of a single bond through a bivalentmonoatom linkage which is a part of FRAG. Preferably, the monoatomlinkage is oxy, thio or seleno when FRAG is a chromogen and oxy or thiowhen FRAG is a fluorogen. Most preferably, Q is oxy.

R², R³ and R⁴ in the above quinone structure are independently hydrogen,substituted or unsubstituted alkyl of 1 to 10 carbon atoms (e.g. methyl,ethyl, hydroxymethyl, methoxymethyl, benzyl, etc.) substituted orunsubstituted aryl (e.g. phenyl, naphthyl, methylnaphthyl,p-nitrophenyl, m-methoxyphenyl, phenylsulfonamido, etc.) or an electronwithdrawing group which generally has a positive Hammett sigma value,and preferably has a sigma value greater than about 0.06. Hammett sigmavalues are calculated in accordance with standard procedures described,e.g. in Steric Effects in Organic Chemistry, John Wiley & Sons, Inc.,1956, pp. 570-574 and Progress in Physical Organic Chemistry, Vol. 2,Interscience Publishers, 1964, pp. 333-339. Representative electronwithdrawing groups having positive Hammett sigma values include cyano,carboxy, nitro, halo (e.g. fluoro, bromo, chloro, iodo), trihalomethyl(e.g. trifluoromethyl, trichloromethyl, etc.), trialkylammonium,carbonyl, carbamoyl, sulfonyl, sulfamoyl, esters and others known in theart, or alkyl or aryl groups (defined above) substituted with one ormore of these electron withdrawing groups. Preferred electronwithdrawing groups include p-nitrophenyl, m-nitrophenyl, p-cyanophenyland 2,5-dichlorophenyl. Aryl groups with methoxy or acetamido groups inthe meta position are also useful.

Homologues of R², R³, R⁴ or R⁶ having more carbon atoms than thosedescribed above may also be useful in this invention, but it must beunderstood that such increased hydrophobicity may require additionalwater-compatibilizing substituents over the smaller homologues.

R³ can also be R¹ thereby potentially providing a 2:1 molar ratio ofdetectable species molecules to original RIND compound molecules.

Alternatively, R³ and R⁴, taken together, can represent the carbon atomsnecessary to complete a substituted or unsubstituted strained fusedcarbocyclic ring attached to the quinone nucleus. Ring strain makesreduction potentials more positive, e.g. see Rieke et al, TetrahedranLetters, No. 50, pp. 4381-4384 (1969). For example, such a ring (mono-or bicyclic) can have from 4 to 8 carbon atoms in the backbone, and ispreferably a 5-membered mono- ring or a 7- or 8-membered bicyclic ring.

In the RIND compounds described above, the water-compatibilizing moietyis a part of one or more of R¹, R², R³ or R⁴. Preferably, it is a partof R², or of R⁶ or FRAG which are parts of R¹ defined above.

Representative and preferred RIND compounds of this invention are listedin Table I below in reference to the following structure: ##STR5## TheE_(1/2) values in Table I were determined for the quinone nucleus ofthis structure having a hydrogen atom in place of ##STR6## The E_(1/2)values (where available) were measured in an aqueous solution of thequinone including N,N-dimethylformamide and sodium phosphate buffer (pH7). A standard calomel electrode was used as a standard. The E_(1/2)values were corrected to a normal hydrogen electrode (NHE). E_(1/2)values not available are denoted by "NA".

                                      TABLE I                                     __________________________________________________________________________    RIND                                                      E.sub.1/2 (mV,      Compound                                                                            R.sup.6   R.sup.2   R.sup.4                                                                             R.sup.3                                                                             FRAG                NHE)                __________________________________________________________________________    I.    CH.sub.3                                                                                 ##STR7##                                                                                ##STR8##                                                                                  ##STR9##           +220                II.   "                                                                                        ##STR10##                                                                               ##STR11##  "                   +200                III.  "                                                                                        ##STR12##                                                                               ##STR13##                                                                                 ##STR14##          NA                  IV.   "                                                                                        ##STR15##                                                                               ##STR16##  "                   NA                  V.    "                                                                                        ##STR17##                                                                               ##STR18##  "                   +200                VI.                                                                                  ##STR19##                                                                               ##STR20##                                                                               ##STR21##  "                   +220                VII.  CH.sub.3                                                                                 ##STR22##                                                                               ##STR23##                                                                                 ##STR24##          +220                VIII. "                                                                                        ##STR25##                                                                               ##STR26##  "                   +200                IX.   "                                                                                        ##STR27##                                                                               ##STR28##                                                                                 ##STR29##          +200                X.    "                                                                                        ##STR30##                                                                               ##STR31##                                                                                 ##STR32##          +222                XI.                                                                                  ##STR33##                                                                               ##STR34##                                                                               ##STR35##                                                                                 ##STR36##          +222                __________________________________________________________________________

RIND compound V is preferred in the practice of this invention.

Some water-compatible reducible compounds of this invention can beprepared by the general synthetic procedures described in U.S. Ser. No.824,766, noted above. Others, e.g. RIND Compounds II-V, VII and IX ofTable I, are prepared by novel procedures like those shown in Examples1-3 below, involving oxazine ring opening using manganese dioxide andhydrolysis of the t-butylester group using trifluoroacetic acid.

Other water-compatible RIND compounds useful in the practice of thisinvention include those having the appropriate E_(1/2) values, one ormore water-compatibilizing moieties as described above, and thestructure CAR--R¹)_(n) wherein:

(1) CAR- is a substituted or unsubstituted nucleus of a1,2-naphthoquinone, 1,2-, 1,4- or 9,10-anthraquinone,4,4'-diphenoquinone, azuloquinone or 1,6-[10]-anulenoquinone wherein R¹is attached to the nucleus one carbon atom distant or in the periposition from one of the oxo groups of the nucleus. The nucleus can besubstituted with one or more electron withdrawing groups as describedabove for R² or have one or more strained fused rings as described abovefor R³ and R⁴. R¹ is ##STR37## as defined above, and n is an integer of1 or 2.

(2) CAR- is ##STR38## any of which can be substituted with one or moreelectron withdrawing groups as described above for R², R³ and R⁴. R¹ is##STR39## as defined above, and n is 1 or 2.

(3) CAR- is a substituted or unsubstituted nitrobenzenoid nucleus of thestructure ##STR40## wherein R⁷ is substituted or unsubstituted alkyl of1 to 12 carbon atoms (e.g. methyl, ethyl, methoxymethyl, isopropyl,dodecyl, etc.), and R¹ is ##STR41## as defined above and n is 1. Thesecompounds are similar to some described in U.S. Pat. No. 4,139,379.

All of these reducible compounds can be prepared using techniques andstarting materials known in the art or readily apparent to a skilledsynthetic chemist.

The water-compatible reducible compounds described herein can beprepared as an aqueous composition comprising a suitable buffer, andoptionally a water-miscible solvent.

A composition can be prepared in the following general manner with theparticular details of such a preparation illustrated in Example 4 below.The reducible compound is dissolved in the buffer at a concentrationwhich depends upon its molecular weight, but generally at from about 1to about 100, and preferably from about 5 to about 80, mg per ml ofbuffer. Alternatively, the reducible compound can be dissolved in thewater-miscible solvent and then mixed with a suitable buffer.

The buffer generally maintains the assay at physiological pH (9 orless). The concentration of buffer in the aqueous composition can varywidely, but is generally from about 0.01 to about 0.1 molar.Representative buffers include phosphates, borates and others reportedby Good et al in Biochemistry, 5, 467 (1966), and Anal. Biochem., 104,300 (1980).

The water-compatible reducible compounds described herein are useful incompositions for analytical determination (i.e. qualitative orquantitative detection) of aqueous and nonaqueous liquids, e.g.biological fluids, manufacturing processes, wastewater, food stuffs,etc. Determinations can be made of various analytes via a singlereaction or sequence of reactions which bring about reduction of thecompound and release of the detectable species. The analytes includeliving cells (e.g. bacteria, white blood cells, yeast, fungi, etc.),enzymes (e.g. oxidoreductases such as lactate dehydrogenase, pyruvatedehydrogenase, glucose-6-phosphate dehydrogenase, etc., oxidases such asglucose oxidase, lactate oxidase, α-glycerophosphate oxidase, etc.,transferases such as alanine aminotransferase, aspartateaminotransferase, etc., hydrolases such as lipase, carboxyesterase, etc.and other NADH-based, FADH-based or oxidase-based assays), biological orchemical reductants other than living cells which will reduce thereducible compound (e.g. ascorbates, cysteine, glutathione, thioredoxin,etc.), metabolizable substances (e.g. glucose, lactic acid,triglycerides, cholesterol, etc.), immunoreactants (e.g. antigens,antibodies, haptens, etc.).

The compositions can be used to monitor enzyme redox reactions as wellas flavin adenine dinucleotide (FAD-FADH)-based and nicotinamide adeninedinucleotide (NAD-NADH)-based and (NADP-NADPH)-based reactions. In suchinstances, the reducible compound can be used to provide a detectablespecies in place of NADH, FADH or NADPH.

The present invention is particularly useful in detecting or quantifyingliving cells in biological samples. Although any biological samplesuspected of having living cells therein (e.g. food, tissue, groundwater, cooling water, pharmaceutical products, sewage, etc.) can beanalyzed for bacteria, yeast, fungi, etc. by this invention, theinvention is particularly useful for bacterial detection in aqueousliquids, such as human and animal fluids (e.g. urine, cerebral spinalfluid, blood and the like as well as stool secretions) and suspensionsof human or animal tissue. The practice of this invention isparticularly important for detection of urinary tract infections inurine (diluted or undiluted).

When determining living cells using the reducible compounds, it ispreferable for rapid dye release in such determinations that the livingcells interact with an electron transfer agent (herein ETA). Thepresence of an ETA may also provide more efficient dye release foranalytical determinations of nonliving analytes. The ETA is a mobilecompound which acts as an intermediary between the substance beingdetermined (e.g. living cell) and the reducible compound. Some ETAs maywork better than others in determining a given organism (see e.g.Example 11 below).

In general, the ETA compounds useful in the practice of this inventionhave an E_(1/2) in the range of from about -320 to about +400 mV asmeasured in aqueous buffer (pH 7) versus the normal hydrogen electrodeusing a differential pulse polarographic technique with a PARPotentiostat (Princeton Applied Research, Princeton, N.J.). In general,the potential of the ETA should be more positive than the potential ofthe substance to be determined (i.e. analyte) and less positive than thepotential of the reducible compound. That is, the ETA should be moreeasily reduced than the analyte and less easily reduced than thereducible compound. They are generally present at a concentration thatis dependant upon the concentration of the analyte, and preferably at aconcentration of from about 1×10⁻⁷ molar to about 1×10⁻³ molar.

ETA compounds useful in the practice of this invention include phenazinemethosulfate, phenazine ethosulfate and similar compounds known to oneskilled in the art. Combinations of different ETA compounds can be usedif desired.

Preferred ETA compounds useful in the practice of this invention whichprovide further advantages of low background are those which are thesubject of copending and commonly assigned U.S. Ser. No. 699,374 filedby Mura et al Feb. 7, 1985. In general, those compounds are substitutedbenzo- and naphthoquinones. Examples of this class of quinones include2,3-dimethyl-5-hydroxymethyl-1,4-benzoquinone,2,5-dimethoxy-1,4-benzoquinone, 2,3,5-trimethyl-1,4-benzoquinone,tetramethyl-1,4-benzoquinone, 2,3-dimethoxy-5-methyl-1,4-benzoquinone,2-hydroxymethyl-1,4-naphthoquinone and2-(2-hydroxyethyl)-1,4-naphthoquinone.

The detection of living cells, and particularly of microorganisms, isoften carried out in the presence of a nutrient for those cells althoughits presence is not essential. Any nutrient media can be used whichcontains useful carbon, and optionally nitrogen, sources. Suitablenutrient media having proper components and pH are well known in theart. Particularly useful nutrients are glucose or tryptose alone or incombination.

The present invention is adaptable to either solution or dry assays. Ina solution assay, a solution containing a reducible compound, andpreferably an ETA, is prepared and contacted and mixed with a liquidtest sample suspected of containing the living cells or analyte to bedetermined. The ETA can also be mixed with the test sample prior tomixing with the reducible compound. Generally the reducible compound ismixed with the test sample in a suitable container (e.g. test tube,petri dish beaker, cuvette, etc.). The resulting solution is gentlymixed and incubated for a relatively short time (i.e. up to about 30minutes) at a temperature up to about 40° C., and generally from about20° to about 40° C. The test sample is then evaluated by measuring thedetectable species, for example, at a wavelength in the spectralabsorption band of a chromagen species, or at a wavelength in theemission band of a fluorogen species which band is different than theband the reducible compound had prior to species release. Such anevaluation can be done with suitable detection equipment.

A pretreatment step to remove interferents or to concentrate cells canalso be carried out before the assay, if desired.

A solution assay can also be carried out by contacting a porous,absorbent material, e.g. paper strip, containing the test sample with asolution of the reducible compound. The analyte in the test sample canmigrate from the porous material into the solution and initiate theanalytical reactions needed for determination. In solution assays, theamount of water-compatible reducible compound present is at least about0.001, and preferably from about 0.01 to about 1.0, millimolar. Otherreagents can be present in amounts readily determined by one skilled inthe art.

Alternatively, the method of this invention can be practiced in a dryassay with a dry analytical element. Such an element can be a absorbentcarrier material, i.e. a thin sheet or strip of self-supportingabsorbent or bibulous material, such as filter paper or strips, whichcontains the reducible compound or a dried residue of the dispersioncomprising same. such elements are known in the art as test strips,diagnostic elements, dip sticks, diagnostic agents and the like.

When employed in dry analytical elements, the reducible compoundsdescribed herein can be incorporated into a suitable absorbent carriermaterial by imbibition or impregnation, or can be coated on a suitableabsorbent carrier material. Alternatively, they can be added to theelement during an assay. Useful carrier materials are insoluble andmaintain their structural integrity when exposed to water orphysiological fluids such as urine or serum. Useful carrier materialscan be prepared from paper, porous particulate structures, cellulose,porous polymeric films, glass fiber, woven and nonwoven fabrics(synthetic and nonsynthetic) and the like. Useful materials andprocedures for making such elements are well known in the art asexemplified by U.S. Pat. Nos. 3,092,465 (issued June 4, 1963 to Adams etal), 3,802,842 (issued Apr. 9, 1974 to Lange et al), 3,915,647 (issuedOct. 28, 1975 to Wright), 3,917,453 (issued Nov. 4, 1975 to Milligan etal), 3,936,357 (issued Feb. 3, 1976 to Milligan et al), 4,248,829(issued Feb. 3, 1981 to Kitajima et al), 4,255,384 (issued Mar. 10, 1981Kitajima et al), and 4,270,920 (issued June 2, 1981 to Kondo et al).

A dry assay can be practiced to particular advantage with an analyticalelement comprising a support having thereon at least one porousspreading zone as the absorbent carrier material. The reducible compoundcan be in the spreading zone or in a different zone (e.g. reagent zone,registration zone, hydrophilic zone, etc.). The spreading zone can beprepared from any suitable fibrous or non-fibrous material or mixturesof either or both.

The spreading zone can be prepared using fibrous materials, either mixedwith a suitable binder material or woven into a fabric, as described inU.S. Pat. No. 4,292,272 (issued Sept. 29, 1981 to Kitajima et al), frompolymeric compositions (e.g. blush polymers) or particulate materials,with or without binding adhesives, as described in U.S. Pat. Nos.3,992,158 (issued Nov. 16, 1976 to Przybylowicz et al), 4,258,001(issued Mar. 24, 1981 to Pierce et al) and 4,430,436 (issued Feb. 7,1984 to Koyama et al) and Japanese Patent Publication 57(1982)-101760(published June 24, 1982). It is desirable that the spreading zones beisotropically porous, meaning that the porosity is the same in eachdirection in the zone as created by interconnected spaces or poresbetween particles, fibers, polymeric strands, etc.

The dry analytical element of this invention can be a singleself-supporting porous spreading zone containing a reducible compoundand any other desired reagents for a particular use, but preferably suchzone is carried on a suitable support. Such a support can be anysuitable dimensionally stable, and preferably, nonporous and transparent(i.e. radiation transmissive) film or sheet material which transmitselectromagnetic radiation of a wavelength between about 200 and about900 nm. A support of choice for a particular element should becompatible with the intended mode of detection (reflection, fluorescenceor transmission spectroscopy) and inert to chemical reagents and liquidsamples used in the assay. Useful support materials include polystyrene,polyesters [e.g. poly(ethylene terephthalate)], polycarbonates,cellulose esters (e.g. cellulose acetate), etc.

The elements can have more than one zone, e.g. a reagent zone, aregistration zone, subbing zone, etc. The zones are generally in fluidcontact with each other, meaning that fluids, reagents and reactionproducts can pass between superposed regions of adjacent zones.Preferably, the zones are separately coated superposed layers, althoughtwo or more zones can be in a single coated layer. Besides the patentsnoted above, suitable element formats and components are described also,for example, in U.S. Pat. Nos. 4,042,335 (issued Aug. 16, 1977 toClement) and 4,144,306 (noted above) and Reissue 30,267 (reissued May 6,1980 to Brushi).

In the elements of this invention, the amount of the water-compatiblereducible compound can be varied widely, but it is generally present ina coverage of at least about 0.01, and preferably from about 0.05 toabout 0.2, g/m². Optional, but preferred reagents (e.g. ETA, nutrient,buffer, etc.) are generally present in the following coverages:

ETA: generally at least about 0.001, and preferably from about 0.01 toabout 1, g/m²,

nutrient: generally at least about 0.05, and preferably from about 0.1to about 2, g/m² (used only in living cell detection), and

buffer (pH≦9): generally at least about 0.1, and preferably from about0.5 to about 2, g/m².

One or more of the zones can contain a variety of other desirable, butoptional, components, including activators, binders (generallyhydrophilic), antioxidants, etc. as is known in the art, as well as anyreagents needed for assay of a particular analyte.

In one embodiment of this invention, an element for detection oforganisms (e.g. yeast, fungi, bacteria, etc.) in an aqueous liquidcomprises an electron transfer agent and a reducible compound, both ofwhich are described above. It is desirable that these elements alsocontain a nutrient for the living cells and a buffer which maintainsphysiological pH during the assay (e.g. when contacted with a 1-200 μlsample of test liquid). Such an element can be used to detect bacteria,for example, in a urine sample (e.g. one pretreated to eliminateinterferents or to concentrate cells) by physically contacting thesample and element in a suitable manner, and detecting the detectablespecies released from the reducible compound as a result of the presenceof the bacteria at the appropriate wavelength.

In another embodiment of this invention, an element is used for thedetermination of a nonliving biological or chemical analyte in anaqueous liquid. If the analyte is not capable of directly reducing thereducible compound described herein to release a detectable species, theassay also includes the use of an interactive composition comprising oneor more reagents which interact with the analyte to produce a productwhich will reduce the reducible compound. This interactive compositioncan be incorporated into the element or added at the time of the assay.Examples of such analytes are described above. The amount of detectablespecies detected can be correlated to the amount of analyte present inthe liquid sample.

The element of this invention is also useful for determining otherreductants such as ascorbate (ascorbic acid and equivalent alkali metalsalts), cysteine, glutathione, thioredoxin and the like.

A variety of different elements, depending on the method of assay, canbe prepared in accordance with the present invention. Elements can beconfigured in a variety of forms, including elongated tapes of anydesired width, sheets, slides or chips.

The assay of this invention can be manual or automated. In general, inusing the dry elements, an analyte or living cell determination is madeby taking the element from a supply roll, chip packet or other sourceand contacting it with a sample (e.g. 1-200 μl) of the liquid to betested so that the sample mixes with the reagents in the element. Suchcontact can be accomplished in any suitable manner, e.g. dipping orimmersing the element into the sample or, preferably, by spotting theelement by hand or machine with one or more drops of the sample with asuitable dispensing means.

After sample application, the element is exposed to any conditioning,such as incubation, heating or the like, that may be desirable toquicken or otherwise facilitate obtaining any test result.

Detection of an analyte or living cell is achieved when thewater-compatible reducible compound is reduced releasing a species whichcan be detected in a suitable manner. Preferably, as noted above, thedetectable species is a colorimetric dye or fluorescent dye which can bedetected with standard colorimetric or fluorometric apparatus anddetection procedures. If the detectable species is other than achromogen or fluorogen, for example, a chemiluminescent orphosphorescent moiety, suitable chemiluminescence or phosphorescencedetecting means can be employed. Spectral determinations can be made atthe maximum wavelength of the detectable species or at wavelengths otherthan the maximum wavelength.

Reagents used in the following examples were obtained as follows:N-2-hydroxyethylpiperizine-N'-2-ethanesulfonic acid (HEPES) buffer andphenazine methosulfate from Sigma Chemical Co. (St. Louis, Mo., U.S.A.),brain heart infusion broth (BHI) and Sabaroud's dextrose (SAB) brothfrom Difco Labs (Detroit, Mich., U.S.A.), TRITON X-100 surfactant fromRohm & Haas (Philadelphia, Pa., U.S.A.), and the bacterialmicroorganisms from the American Type Culture Collection (ATCC) inRockville, Md., U.S.A.. All other reagents were either obtained fromEastman Kodak Company (Rochester, N.Y., U.S.A.) or prepared using knownstarting materials and procedures.

Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923)cells were individually grown overnight in BHI medium at 37° C. withoutshaking and transferred daily. Forty ml of the cells were harvested bycentrifugation and resuspended in 10 ml of a 0.05 molar HEPES buffer (pH7.8). The absorbance measured at 620 nm was adjusted to 0.5 and 1.0,respectively. An absorbance of 0.833 (620 nm) was found to be equivalentto about 5×10⁸ cells/ml. Candida albicans (ATCC 14053) cells were grownin SAB broth overnight at 25° C. with agitation, harvested bycentrifugation, washed and resuspended in HEPES buffer. The cellsuspension was adjusted to an optical density of about 1.0.

In the preparation of the illustrated reducible compounds, the identityand purity of the intermediates were determined by infrared (IR) spectraas measured in a commercially available Perkin-Elmer 137spectrophotometer [sharp(s) or broad(b) bands yielding structuralinformation are reported in reciprocal centimeters (cm⁻¹)], by nuclearmagnetic resonance (NMR) spectra measured in a standard Perkin-Elmer NMRspectrophotometer [chemical shifts reported in δ values in ppm totetramethylsilane at broad(b), singlet(s), multiplet(m) or broadsinglets(bs) peaks], or by field desorption mass spectral analysis(FDMS) measured in a Varian MAT 731 Spectrophotometer. The identity andpurity of final products were determined by IR, NMR spectroscopy FDMS,and elemental analysis.

The following examples are presented to illustrate the practice of thisinvention. The identified RIND compounds are the reducible compoundsincluded in Table I above.

EXAMPLE 1 Preparation of RIND Compound I

The Intermediates A'-F' for making RIND Compound I of Table I above wereprepared according to the procedures of Steps 1-6 of Example 1 of U.S.Ser. No. 824,766, noted above, using p-cyanoaniline as a startingmaterial instead of p-nitroaniline in Step 1.

The carbamoyl chloride from Step 6 was converted to Intermediate G' bythe following procedure: carbamoyl chloride (15.8 g, 40 mmole) was addedto a solution of t-butyl-5-hydroxy-2-nitrobenzoate (8 g, 33 mmole) andN,N-dimethylaminopyridine (catalytic amount) in pyridine (150 ml) andthe resulting mixture was stirred for 18 hours under a nitrogenatmosphere in the dark. The reaction mixture was then poured into dilutehydrochloric acid/ice water (2 liters) and the precipitated yellow solidisolated by filtration, washed with water and air dried. Columnchromatography (silica, 97:3, dichloromethane:ether) gave 19 g (96%yield) of Intermediate G' as a yellow foam. Analysis confirmed thestructure: IR (KBr) cm⁻¹, 2250 (w, CN), 1725 (bs, --CO--), 1650 (s,quinone), NMR (CDCl₃) δ, 1.2-2.0 (m, bicyclic H's), 1.5 (s, t-butyl),2.8-3.0 (d, NCH₃), 3.25-3.5 (bd, CH₂ --N), 4.3-4.55 (bd, bridgehead),7.15-8.0 (m, ArH's).

Intermediate G' was then converted to RIND Compound I by the followingprocedure. A solution of Intermediate G' (19 g, 31.8 mmole) andtrifluoroacetic acid (75 ml) in dichloromethane (200 ml) was stirredunder a nitrogen atmosphere in a dark area for 2.25 hours. The reactionmixture was poured into ice water and the layers were separated. Theorganic layer was washed with cold water, dried (sodium sulfate) and thesolvent removed to give a yellow foam. This material was filteredthrough silica using 95:5 dichloromethane:ether to give 10 g (58% yield)of RIND Compound I. IR (KBr) cm⁻¹, 2250 (w, CN), 1730 (bs, --CO--), 1650(s, quinone). NMR (CDCl₃) δ 1.2-2.0 (m, bicyclic H's), 2.7-3.1 (d,NCH₃), 3.35-3.36 (m, bridgehead H's), 4.3-4.55 (d, --CH₂ --N), 6.9-8.0(m, ArH's). Elemental analysis also confirmed the structure.

EXAMPLE 2 Preparation of RIND Compound V

Intermediates A'-D' for the preparation of RIND Compound V of Table Iabove were prepared according to the procedures of Steps 1-4 of Example1 of U.S. Ser. No. 824,766, noted above, using 4-amino-t-butylbenzoateas a starting material instead of p-nitroaniline in Step 1.

Intermediate D' was converted to Intermediate E' by the following novelprocedure: a suspension of Intermediate D' (20 g, 47.4 mmole) andmanganese dioxide, activated (Aldrich Chemical Co., Milwaukee, Wis.,U.S.A., 40 g, 474 mmole) in dicloromethane (1 liter) was stirred for 1hour at 25° C. The mixture was filtered through a celite pad and thefiltrate was concentrated under reduced pressure to give 19 g of thedesired amine intermediate. NMR (CDCl₃), δ 1.1-1.9 (m, bicyclic ringH's), 1.6 (s, t-butyl), 1.85 (s, CH₃ CN), 3.1-3.45 (m, bridgehead andCH₂ N), 7.1 and 8.1 (AA'XX', J=9 cps, ArH's).

Intermediate E' was converted to Intermediate F' by the procedure ofStep 6 of Example 1 of U.S. Ser. No. 824,766, noted above. IntermediateF' was converted to Intermediate G' by the procedure of Step G ofExample 21 of U.S. Ser. No. 824,766.

Intermediate G' was converted to RIND Compound V using the followingprocedure carried out in the dark. A solution of Intermediate G' (9.1 g,14.4 mmole) and trifluoroacetic acid (40 ml) in methylenedichloride (130ml) was stirred under nitrogen for 2.5 hours. The solvent was removedunder reduced pressure and the residue was dissolved in a minimal amountof acetone. The acetone solution was slowly added to a dilute solutionof hydrochloric acid in ice water (400 ml) and the resulting semisolidwas collected by filtration, washed with water and air dried. Thematerial was recrystallized from ethyl acetate to give 6.3 g of RINDCompound V as a yellow solid. IR (KBr) cm⁻¹, 1730 (bs, --CO--), 1650 (s,quinone --CO--). NMR (CDCl₃) δ 1.1-2.0 (m, bicyclic H's), 2.9 (d, 12ppm, CH₃ N), 3.35 (d, 9 ppm, bridgehead H's), 4.5 (d, 16 ppm, --CH₂ N),6.45-8.6 (m, phenalenone and Ar --COOH). Elemental analysis: Calculatedfor C₃₅ H₂₇ NO₇ : C, 73.3, H, 4.7, N, 2.4. Found: C, 73.2, H, 5.1, N,2.2.

EXAMPLE 3 Preparation of RIND Compound VI

The quinone nucleus was prepared by a standard oxidation of thecorresponding hydroquinone, which had been prepared according to theprocedure described in Steps 1-3 of Example 1 in U.S. Ser. No. 824,766of Belly et al, reference above, using p-cyanoaniline instead ofp-nitroaniline.

This quinone (5.2 g, 18 mmole) was added to a mixture of hydrobromicacid (30% in acetic acid, 48 ml), 37% formalin (18 ml) and acetic acid(140 ml), and the resulting solution was heated at 55° C. for 18 hours.After cooling, the reaction mixture was poured into ice water (500 ml),and the precipitated yellow solid was collected and recrystallized fromethanol to give 2.4 g of the bromomethyl intermediate having a m.p. of201°-202° C. An NMR spectrum confirmed the structure.

A mixture of the bromomethyl intermediate (9.2 g, 24 mmole),p-amino-t-butylbenzoate (9.25 g, 48 mmole) and silver(I)carbonate (6.6g, 24 mmole) in N,N-dimethylformamide (360 ml) was stirred under anitrogen atmosphere in a dark area for 36 hours. The resulting mixturewas poured into dilute hydrochloric acid/ice water (1500 ml) and theprecipitated brown solid was collected by filtration, washed with waterand dried under reduced pressure in a desiccator. Chromatography(silica, 70:30 ligroine:ethyl acetate) afforded the desired amineintermediate, yield of 7 g. Mass spectral analyses, nuclear magneticresonance and infrared spectroscopy confirmed the structure.

The amine intermediate was converted to the corresponding carbamoylchloride by the procedure described in Step 6, Example 1 of the Belly etal application noted above.

A mixture of the carbamoyl chloride (7 g, 12.6 mmole),6-hydroxyphenalenone (2.05 g, 10.4 mmole), prepared by the proceduredescribed in U.S. Ser. No. 824,757 of Babb et al, filed Jan. 31, 1986and entitled BIOLOGICAL AND ANALYTICAL USES OF PHENALENONE ANDBENZPHENALENONE COMPOUNDS, and N,N-dimethylaminopyridine (catalyticamount) in pyridine (75 ml) was stirred under nitrogen in a dark areafor 18 hours. The mixture was poured into dilute hydrochloric acid/icewater and the precipitated solid was collected by filtration, washedwith water and dried in air in a dark area. Chromatography (silica,98:2, dichloromethane:acetone) gave 5 g of the desired intermediate. Ananalytical sample was obtained by recrystallization from ether to give apale yellow solid. NMR (CDCl₃)δ, 1.2-2.0 (m, --CH₂ CH₂ -- of bicyclicring), 1.6 (s, t-butyl), 3.21-3.59 (m, bridgehead H's), 4.9 (s, --CH₂N--), 6.6-6.75 (d, dye H's), 7.1-8.2 (m, dye and aryl H's), 8.6-8.7 (d,dye H's). IR (KBr) cm⁻¹, 2240 (s, CN), 1720 (b, C═O), 1640 (s, quinone).

A solution of the t-butylcarboxyphenyl intermediate (2 g, 2.8 mmole) andtrifluoroacetic acid (10 ml) in dichloromethane (25 ml) was stirredunder a nitrogen atmosphere in a dark area for 2 hours. The reactionsolution was concentrated in a dark area to remove the solvent and theresidue was dissolved in tetrahydrofuran and the solution was pouredinto dilute hydrochloric acid/ice water. The precipitated solid wascollected by filtration, washed with water and dried in a dark area.Column chromatography (silica, 89.5 dichloromethane, 10 acetone, 0.5acetic acid) afforded 1.4 g of RIND Compound VI of Table I above as ayellow solid. IR (KBr, cm⁻¹), 2250 (w, CN), 1720 (bs, C═O), 1645 (s,quinone). NMR (CDCl₃)δ, 1.15-2.0 (m, bicyclic H's), 3.2-3.5 (d,bridgehead H's), 4.95 (s, CH₂ N), 6.6-8.7 (m, ArH's). Elemental analysisalso confirmed the structure.

EXAMPLE 4 Preparation of Aqueous Composition

Compositions of this invention were prepared as follows: the appropriateRIND compound was dissolved in 250 ml of N,N-dimethylformamide (DMF),which had been acidified by the addition of 0.1% concentrated sulfuricacid, and the resulting solution was added to 25 ml of 0.05 molarpotassium phosphate buffer or 0.05 molar HEPES buffer (pH 7.8).

EXAMPLES 5 AND 6 Assay for E. coli: Comparison of Water-Compatible RINDCompounds With and Without Surfactant

This example uses two water-compatible RIND Compounds of this invention(I and X of Table I above) in a comparison of an assay of the presentinvention using a composition without surfactant to an assay of Belly etal U.S. Ser. No. 824,766 using a composition including a surfactant.

E. coli cells were grown overnight in BHI medium without shaking at 37°C. Forty milliliters of cells were harvested by centrifugation. Theresulting pellet was resuspended in 25 ml potassium phosphate buffer (pH7.5, μ =0.05) and the resulting suspension was centrifuged. The pelletwas washed and resuspended in 25 ml of buffer, and an aliquot of thesuspension was diluted with the same buffer to obtain an absorbance of0.1 at 620 nm.

A. Compositions of the present invention were prepared from thefollowing solutions: phenazine methosulfate (PMS) or2-hydroxymethyl-5,6-dimethyl-1,4-benzoquinone (HDMBQ) (9.8×10⁻³ molar inmethanol), RIND Compounds I or X (1.7×10⁻² molar inN,N-dimethylformamide, DMF), and glucose solution (10% in water).

A 1 cm pathlength cuvette (4 ml capacity) was filled with 0.1 ml cellsuspension, (final concentration 6×10⁻⁷ cells/ml), 3.0 ml phosphatebuffer, 50 μl glucose solution (final concentration 8.8×10⁻² molar), and25 μl of either PMS or HDMBQ (final concentration 7.7×10⁻⁵ molar). Thecuvette was sealed to prevent evaporation and thermally equilibrated at37° C. The reaction was initiated by the addition of 14 ml of RINDCompound I or X (final concentration 7.6×10⁻⁵ molar). Control solutionswere prepared without any cells.

B. Compositions of the prior art using TRITON X-100 surfactant wereprepared from the following solutions: (1) PMS or HDMBQ (9.8×10⁻³ molarin methanol), (2) RIND Compound I or X (7.1×10⁻² molar in DMF), (3) 50μl of solution 2 plus 100 μl TRITON X-100 surfactant plus 5 ml phosphatebuffer, (4) glucose solution (10% in water), and (5) 6×10⁹ cells/ml inphosphate buffer.

A 1 cm pathlength cuvette was filled with 188 ml of solution 3, finalRIND concentration 7.6×10⁻⁵ molar, 6 μl of solution 4, final glucoseconcentration 8.8×10⁻³ molar, and 12.5 μl of solution 5, final cellconcentration 6×10⁻⁷ cells/ml. The cuvette was covered and thermallyequilibrated at 37° C. The reaction was then initiated by the additionof 3 μl of solution 1, final ETA concentration 7.7×10⁻⁵ molar. Controlsolutions were prepared without any cells.

Reactions ere followed by monitoring the appearance of dye at 402 nm.The results, shown in Table II below, are averages of 3 or 4determinations and indicate the increased cell response fromcompositions of this invention.

                  TABLE II                                                        ______________________________________                                        RIND                % Dye Released After 10 Minutes                           Compound ETA        With E. Coli.:                                                                           Without E. Coli.:                              ______________________________________                                        I        PMS        60         1.3                                            I (Control)                                                                            "          49         0.7                                            I        HDMBQ      53         0.8                                            I (Control)                                                                            "          31         0.1                                            X        PMS        83         1.2                                            X (Control)                                                                            "          56         1.1                                            X        HDMBQ      86         1.4                                            X (Control)                                                                            "          65         0.7                                            ______________________________________                                    

EXAMPLE 7 Comparative Assays Using Water-Compatible RIND andWater-Incompatible RIND Compounds

Assays were run in a Milliliter HA 96 Well Filtration Plate (MilliporeCorp., Bedford, Mass., U.S.A.). Fluorescence was measured using aDynatech Microfluor Reader (Dynatech Laboratories, Alexandria, Va.,U.S.A.) modified to read at excitation 510 nm and emission 620 nm.

A Control composition was prepared as follows: 250 μl of RIND solution(16 mg of Control RIND compound/ml acidified DMF), 500 μl TRITON X-100surfactant solution, 25 ml HEPES buffer (pH 7.8), 500 μltrimethyl-1,4-benzoquinone (TMBQ) (1.5 mg/ml methanol), and 500 μlglucose solution (10% solution in water). The Control RIND compound waslike Compound V of Table I above except that it has a cyano group inplace of the carboxy group.

A composition of this invention was prepared as follows: 250 μl of RINDV (16 mg/ml of acidified DMF), 25 μl HEPES buffer, 500 μl TMBQ (1.5mg/ml methanol) and 500 ml glucose solution (10% in water).

The assays were carried out in duplicate by adding 150 μl of S. aureuscell suspensions (final concentration 1×10⁻⁷ cells/ml) to the wells,warming the plate to 37° C. and adding 150 μl of the control and testcompositions. Blank solutions were prepared without any cells.Fluorescence was then measured at zero time and after 30 minutesincubation at 37° C. Data are shown in Table III below. These dataindicate that the present invention provides increased release of dye inthe absence of a surfactant.

                  TABLE III                                                       ______________________________________                                                   Δ Relative Fluorescence, 30 minutes                                     Blank      S. Aureus                                               ______________________________________                                        Control      155           2787                                               Test         133          >4000                                               ______________________________________                                    

EXAMPLE 8 Comparison of Cell Response with Water-Compatible RINDCompounds With and Without Surfactant

This example illustrates the increased cell response in compositions ofthis invention using two water-compatible RIND compounds and fourelectron transfer agents.

RIND compounds used were VI and XI from Table I above. ETA's used were:ETA 1, tetramethyl-1,4-benzoquinone, ETA 2,2,3,5-trimethyl-1,4-benzoquinone, ETA 3,2,3-dimethoxy-5-methyl-1,4-benzoquinone, ETA 4,2-hydroxymethyl-1,4-naphthoquinone.

Solutions were prepared from each water-compatible RIND compound andeach ETA in microtitration plates. Control solutions (with TRITON X-100surfactant) contained the following: 9.3 ml HEPES buffer (pH 7.8), 0.1μl RIND compound (final concentration 7.6×10⁻⁵ molar in acidified DMF),0.2 μl TRITON X-100 surfactant, 0.2 μl 10% glucose solution, 0.2 μl ETA(final concentration 3.85×10⁻⁵ molar in methanol) and 0.15 μl E. colicells (final concentration 10⁷ cells/ml HEPES buffer). Test solutionswere prepared from the same components, except no TRITON X-100surfactant was present. Blank solutions were prepared containing allcomponents except cells for background measurements. Fluorescence wasthen measured at zero time and after 30 minutes incubation at 37° C.Data are shown in Table IV below. These data indicate that more dye isreleased in the absence of surfactant in all tests.

                                      TABLE IV                                    __________________________________________________________________________    Δ Relative Fluorescence                                                 (minus background), 30 Minutes                                                ETA 1           2           3           4                                     RIND                                                                               VI    XI    VI    XI    VI    XI    VI    IX                             __________________________________________________________________________    Surfac-                                                                           -  +  -  +  -  +  -  +  -  +  -  +  -  +  -  +                            tant                                                                          Δ                                                                           724                                                                              407                                                                              731                                                                              580                                                                              1390                                                                             876                                                                              1303                                                                             1122                                                                             1281                                                                             317                                                                              1475                                                                             1005                                                                             1886                                                                             1389                                                                             1528                                                                             1328                         Rel.                                                                          Fluor.                                                                        __________________________________________________________________________

EXAMPLE 9 Solution Assay of White Blood Cells

This example demonstrates the use of the present invention to determinewhite blood cells in a solution assay. It also compares a similar assaycarried out with a composition of U.S. Ser. No. 824,766, identifiedabove.

A composition of this invention was prepared as described above inExample 7. Control A composition was prepared similarly to the Controlcomposition of Example 7 whereas Control B was the same as Control Aexcept that the RIND compound used was a water-compatible compound ofthis invention (RIND V). Both Controls A and B contained a surfactant.

Blood samples (8.5 ml) were collected in standard blood collection tubescontaining 1.5 ml of acid citrate dextrose. A 6% dextran solution wasadded to each at a volume of from 1.5 to 2 ml per tube, and the tubecontents were mixed by inversion and allowed to settle for 1.5 hours.The plasma layer from each tube was transferred to sterile 15 mlcentrifuge tubes and 0.5 molar potassium phosphate buffer (pH 7.5)containing 8.9 g/l NaCl was added. The tubes and their contents werethen centrifuged at 1000 rpm for 10 minutes and the supernatantdecanted. The resulting cell pellet was resuspended in 10 ml lysingsolution (8.3 g ammonium chloride, 1 g sodium carbonate and 0.03 gsodium ethylenediaminetetraacetic acid per liter), and then the tubeswere allowed to stand for 5 minutes to allow the solution to clear. Thetube contents were again centrifuged at 1000 rpm for 10 minutes, and thesupernatant decanted. Again, the resulting pellet was resuspended inphosphate buffered saline, centrifuged, the supernatant decanted and thepellet resuspended in 0.5 ml phosphate buffered saline.

White blood cells were counted after preparation by standard proceduresusing a commercially available Coulter Counter instrument equipped witha 30 ml aperture. The counts were corrected for coincidence. The cellswere refrigerated overnight prior to evaluation and then diluted 25 foldin phosphate buffered saline prior to use.

To microliter plate wells were added diluted white blood cell suspension(150 μl at 2.1×10⁵ cells/ml), a RIND compound composition (150 μl),trimethylbenzoquinone ETA (25 μl of 1.5 mg/ml methanolic solution) andglucose (25 μl of 10% w/v). The plates were incubated at 37° C. and thedye released was evaluated by measuring the fluorescence (emission, 620nm, excitation, 540 nm) using a commercially available fluorometerequipped with a Xenon arc lamp source. Three replicates of each testwere made.

The results of the assays, shown in Table V below, indicate that whiteblood cells can only be detected using a water-compatible RIND compoundin a composition lacking a surfactant.

                  TABLE V                                                         ______________________________________                                                     Δ Relative Fluorescence                                    Composition  After 30 Minutes                                                 ______________________________________                                        Control A    -22                                                              Control B     -8                                                              Example 9     52                                                              ______________________________________                                    

EXAMPLE 10 Determination of Bacteria and Yeast With a Dye Element Format

This example illustrates the determination of two species of bacteria,E. coli and S. aureus and a yeast C. albicans using a dry elementcontaining RIND compound VI of Table I above.

Strips of Whatman 3 mm chromatography paper (VWR Scientific, Rochester,N.Y., U.S.A.) were immersed in the following solution: 7 ml methanol, 1ml RIND solution (RIND VI, 0.028 molar in N,N-dimethylformamidecontaining 0.1% sulfuric acid), 1 ml ETA solution(2,3-dimethoxy-5-methyl-1,4-benzoquinone, 0.01 molar methanol) and 1 mlglucose solution (10% solution in water). The strips were then allowedto dry at 25° C. for 1 hour in a dark area.

A standard paper punch was used to cut discs [about 1/4 inch (0.6 cm) indiameter] from the dried strips and the discs were placed in CorningCell Wells™. (Corning Glass Works, Corning, N.Y., U.S.A.).

Ten microliter samples of controls (containing only buffer) and testcell suspensions (E. coli, ˜5×10⁸ cells/ml, S. aureus, ˜5×10⁸ cells/ml,and C. albicans, ˜5×10⁶ cells/ml) were spotted onto the paper discs andfluorescence (excitation 540 nm, emission 620 nm) was measured at zerotime and after 30 minutes of incubation at 37° C. using a DynatechMicrofluor Reader (Dynatech Labs, Alexandria, Va., U.S.A.). The resultsare shown in Table VI below as the change in relative fluorescence after30 minutes. The average of two readings is listed. It is apparent thatthe present invention can be used to determine microorganisms with a dryelement.

                  TABLE VI                                                        ______________________________________                                                  Δ Relative Fluorescence,                                                30 Minutes, 37° C.                                           ______________________________________                                        Control      278                                                              E. coli     2689                                                              S. aureus   2570                                                              C. albicans 2054                                                              ______________________________________                                    

EXAMPLE 11 Assays for Various Organisms Using a Water-Compatible RINDCompound

This example illustrates the use of a reducible compound of thisinvention (RIND I of Table I above) for the determination of severalorganisms. All of the cells were grown as described above, withoutshaking, at 37° C., except Micrococcus luteus and Bacillus subtiliswhich were grown at 30° C., and Pseudomonas aeruginosa which was grownwith shaking.

The assays were carried out as described in Examples 5 and 6 above. Twodifferent ETAs, PMS and HDMBQ (both identified in Examples 5 and 6above), were used. The resulting data are presented in Table VII below.

                  TABLE VII                                                       ______________________________________                                                                          % Dye                                                     Final Cell          Released                                                  concentration       After                                       Organism      (cells/ml) ETA      10 minutes                                  ______________________________________                                        Bacillus subtilis                                                                           1.7 × 10.sup.7                                                                     HDMBQ    99                                          (ATCC 21777)             PMS      79                                          Micrococcus luteus                                                                          1.5 × 10.sup.7                                                                     HDMBQ    62                                          (ATCC 4698)              PMS      59                                          Proteus vulgaris                                                                            9.8 × 10.sup.7                                                                     HDMBQ    68                                          (ATCC 13315)             PMS      67                                          Pseudomonas aeruginosa                                                                      3.5 × 10.sup.7                                                                     HDMBQ    68                                          (ATCC 27853)             PMS       3                                          Serratia marcescens                                                                         2.2 × 10.sup.8                                                                     HDMBQ    12                                          (ATCC 8100)              PMS      21                                          Staphyloccus aureus                                                                         7.6 × 10.sup.7                                                                     HDMBQ    61                                          (ATCC 25923)             PMS      50                                          Streptococcus facaelis                                                                        1 × 10.sup.8                                                                     HDMBQ    41                                          (ATCC 33186)             PMS      20                                          Salmonella typhimurium                                                                      1.4 × 10.sup.8                                                                     HDMBQ    41                                          (ATCC 14028)             PMS      54                                          Klebsiella pneumoniae                                                                       9.8 × 10.sup.7                                                                     HDMBQ    37                                          (ATCC 13883)             PMS      59                                          ______________________________________                                    

The present invention has been described in detail with particularreference to preferred embodiments thereof, but it will be understoodthat variations and modifications can be effected within the spirit andscope of the invention.

We claim:
 1. A method for the determination of an analyte comprising thesteps of:A. contacting a sample of a liquid suspected of containing ananalyte with a water-compatible reducible compound of the structureCAR--R¹)_(n) wherein CAR- is a substituted or unsubstituted aromatic orquinone nucleus, R¹ is a moiety which comprises a shiftable detectablespecies, and n is 1 or 2, provided said compound is constructed suchthat it is capable of being reduced at said pH to release said shiftabledetectable species, and said compound comprises at least onewater-compatibilizing moiety which has a hydrophobic parameter less thanabout -2.0, and further provided that when R¹ is replaced with H,CAR--H)_(n) is constructed such that it has an E_(1/2) of at least about+100 mV when measured in water, and B. detecting the detectable speciesreleased as a result of the presence of said analyte.
 2. The method ofclaim 1 wherein said reducible compound is of the structure CAR- R¹wherein CAR- is ##STR42## R¹ is ##STR43## R² and R⁴ are independentlyhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted aryl or an electron withdrawing group,R³ is R¹, hydrogen,substituted or unsubstituted alkyl, substituted or unsubstituted aryl oran electron withdrawing group, provided at least one of R², R³ and R⁴ isan electron withdrawing group, or R³ and R⁴, taken together, representthe atoms necessary to complete a substituted or unsubstituted strainedfused carbocyclic ring, R⁵ is substituted or unsubstituted alkylene of 1or 2 carbon atoms, R⁶ is substituted or unsubstituted alkyl, substitutedor unsubstituted cycloalkyl or substituted or unsubstituted aryl, Q iscarbonyl or thiocarbonyl, FRAG is a shiftable detectable species whichprovides a detectable species when released from said reduciblecompound, and m is 0 or 1, provided that when R¹ is replaced with H,CAR- H has an E_(1/2) of at least about +100 mV when measured in water,and further provided that R¹, R², R³ or R⁴ comprises saidwater-compatibilizing moiety, and B. detecting the detectable speciesreleased as a result of the presence of said analyte.
 3. The method ofclaim 1 for the determination of a nonliving analyte in the presence ofan interactive composition for said analyte.
 4. The method of claim 1for the determination of living cells in the presence of an electrontransfer agent and a nutrient.
 5. The method of claim 4 for thedetermination of microorganisms.